Diversity of lactic acid bacterial in inasua fermentation.

Background and Objectives
Inasua is one of the traditional fermented fish products in Maluku, Indonesia. There are two types of inasua, i.e. with and without sap. The research aimed to study the succession of lactic acid bacteria (LAB) during fermentation and microbial composition in inasua.


Materials and Methods
The sample of inasua was taken from two traditional producers in Layeni village, Ceram Island. The diversity of lactic acid bacteria was analyzed based on the 16S rRNA gene sequence.


Results
The succession of lactic acid bacteria was strongly influenced by the physicochemical characteristics during fermentation. Lactobacillus plantarum was found dominant in both inasuas fermentation processes. At end of fermentation, L. plantarum was still found dominant in inasua with sap while inasua without sap was dominated by Leuconostoc mesenteroides. In addition, Lactobacillus paracasei (LAB) was found only in inasua with sap. The result of Denaturing Gradient Gel Electrophoresis (DGGE) revealed that Lactobacillus was the dominant bacteria in inasua with sap while Staphylococcus was dominant in inasua without sap.


Conclusion
Inasua with sap was found with higher bacterial diversity index and lower evenness and dominance indices, as well as more complex LAB succession pattern during fermentation and bacterial composition, as opposed to inasua without sap.


INTRODUCTION
Fish is the main source of protein consumed by people in Maluku. Fish is often caught abundantly, making preservation necessary. One of the fish preservation techniques with fermentation is inasua. It is a local wisdom of Teon, Nila, and Serua commu-nities. In addition to being used as a food reserve during lean times (1) in the past, the fermented product was also used by communities as a food stock during shipping when they sold cloves to other islands. Raw materials used to produce inasua are reef fish, salt and coconut sap. However, in certain conditions, producing inasua does not always use coconut sap. While inasua with coconut sap (inasua-S) uses both coconut sap and salt as preservatives, inasua without coconut sap (inasua-NS) uses only salt. Both have distinct sensory characteristics and different shelf life.
Sensory characteristics and shelf life of a fermented product are strongly influenced by microbial diversity. Inasua fermentation occurs spontaneously and involves various types of microorganisms. The main microbe involved in fish fermentation is LAB (2). The bacteria belong to the generally recognized as safe (GRAS) category, making it safe to be in a food product (3). The composition of LAB in traditional fish fermentation is highly determined by the type of carbohydrate and the amount of salt added (4). LAB found in inasua fermentation can be developed as a starter in various fermented products.
DGGE is one of the methods for detecting microbial diversity in a fermented product (5). This method can determine the safety aspects of inasua. The objective of this research was to study the succession of LAB during fermentation and microbial composition in inasua-S and inasua-NS.

MATERIALS AND METHODS
Inasua sampling. The sample of inasua was taken from two traditional producers in Layeni village, TNS Waipia Sub-District, Ceram Island (each produced both inasua-S and inasua-NS). A total of 5 kg brown stripe red snapper (Lutjanus vitta L) was obtained from the sea around the Ceram Island and 2.5 kg was processed into inasua-NS by adding salt only and the remaining 2.5 kg into inasua-S by adding both salt and coconut sap. The inasua was then allowed to ferment in jars at room temperature for 12 weeks. Analysis of LAB succession was carried out in week one until week 12 of fermentation. A 500 g sample from each inasua type was taken into the laboratory for further analysis.
Isolation and characterization of lactic acid bacteria. A 25 g sample from each inasua type was mixed with 225 ml sterile peptone solution and homogenized using stomacher bags. One ml of the homogenized and diluted sample was poured into Petri dishes followed with de Man, Rogosa and Sharp agar (MRSA) media containing 1% CaCO 3 with 3% and 5% and NaCl prior to incubation at room temperature for 48 hours. All isolates obtained were stained with Gram and spore staining, catalase test, and fermentation of carbohydrates (6).

Extraction and amplification of LAB's 16S
rRNA. DNA extraction was carried out following procedure from Presto TM Mini GDNA Kit (Geneaid). The result of DNA extraction was used to amplify 16S rRNA gene. The 16S rRNA gene was amplified using PCR machine with 63F (5'-CAG GCC TAA CAC ATG CAA GTC-3') and 1387R (5'-GGG CGG WGT GTA CAA GGC-3') primers (7). The volume of PCR reaction used was 25 μL, consisting of 12.5 μL GoTaq Green Master Mix 2X (Promega, Madison, W1, USA); 2.5 μL 63F and 1387R primers each (10 pmol); 6.5 μL Nuclease Free Water and 1 µg DNA genome as template. The reaction was amplified in 30 cycles and each PCR comprised pre-denaturation at 95°C for 5 mins, annealing at 55°C for 1 min, elongation at 72°C for 1.5 min, and extension at 72°C for 10 mins. PCR products were visualized using an electrophoresis machine at 80 volts for 45 mins and stained with ethidium bromide.
Construction of phylogeny tree. The amplified DNA was further sequenced and analyzed using ChromasPro software (Technelysium, AU) for sequence coupling. The sequences were then compared with GenBank database using Basic Local Alignment Search Total Nucleotide (BLASTN) software. The obtained homologous sequences were then aligned using MEGA 6.0 software (8) with 1000x bootstrap replications while phylogenetic tree was constructed using Neighbor Joining method. CCCGCCGCGCGCGG-CGGGCGGGG-CGGG-GGCCCGGGGGGACTCCGGGAGGCAGCAG-'3) and P518R (5'-ATTA-CCGC-GGCTGCTGG-'3) primers (9). PCR reaction used a mixture consisting of 1 ml DNA template, 2 μl each primer (20 pmol), 1 μl dNTP (100 mM for each dNTP), 5 μl 10x PCR buffers, 0.25 μl Taq polymerase and 40.7 μl H 2 O. The reaction was amplified in 30 cycles and each PCR comprised initial denaturation at 94°C for 5 mins, denaturation at 94°C for 30 secs, annealing at 55°C for 30 secs, elongation at 72°C for 30 secs, and extension at 72°C for 7 mins. PCR products were visualized using an electrophoresis machine at 80 volts for 45 mins and stained with ethidium bromide.

Analysis of microbial diversity. Diversity index
was analyzed based on the interpretation of CLIQS ID software while relative abundance and Operational Taxonomic Unit (OTU) dominance values of DGGE results using PAST3 software (11). In addition, microbial diversity was analyzed using Shannon-Wiener diversity index obtained based on the OTU richness and the proportion of abundance of each OTU. The formula of Shannon-Wiener index is H'= -Σ (pi log pi). H = diversity index, pi = the proportion of the number of individuals of an OTU to the total number of individual samples in the plot (n/N) (12).

Diversity of culturable lactic acid bacteria.
A total of 50 isolates of lactic acid bacteria were obtained during inasua fermentation, comprising 22 isolates from inasua-NS and 28 isolates from inasua-S. Analysis of gene sequences encoding 16S rRNA from 18 selected isolates (inasua -NS: 6; inasua-S: 12) with GenBank data using the BLAST-N program revealed that all isolates were LAB that are closely related to the Lactobacillus and Leuconostoc groups. The percentage of sequence similarity with target 16S rRNA gene in genBank database was 96-99% (Table 1). The bacteria underwent a succession during fermentation. In inasua-S fermentation, Lactobacillus plantarum and L. rhamnosus were the LAB found at the beginning of fermentation. L. paracasei was found after week 4 of fermentation while Leuconostoc mesenteroides at the end of fermentation. Dominant LAB in inasua-S fermentation, i.e. L. plantarum. Lactobacillus plantarum and L. rhamnosus, were also found at the beginning of inasua-NS fermentation. After week 8 of fermentation, L. mesenteroides was found. L. mesenteroides was also the dominant LAB at the end inasua-NS fermentation (Fig. 1).
Phylogenetic tree analysis revealed that LAB isolates from the fermentation are related with Lactobacillus and Leuconostoc, with the exception of 3 isolates found at the end of inasua-NS fermentation that are closely related to Staphylococcus (Fig. 2).
Metagenomic diversity. The result of the separation of PCR products using DGGE showed that bacterial community pattern based on 16S rRNA gene varied in both samples. The pattern distribution of bacterial community from inasua-NS (4 bands) was less varied than from inasua-S (8 band) in polyacrylamide gel (Fig. 3). Comparison with GenBank database revealed that 4 bands from inasua-NS were identified as Lactobacillus curiae, Staphylococcus pasteuri and two other bands were all S. epidermidis, while 8 bands from inasua-S were identified as L. apinorum, Escherichia fergusonii, L. nagelii, L. paracasei, L. curiae and two other bands were L. hilgardii. The percentage of sequence similarity between DGGE results and target genes in GenBank data was 90-99% (Table 2).
Phylogenetic analysis based on 16S rRNA encoding gene was constructed using neighbor joining model with 1000x bootstrap replications and revealed that the bands found in two types of inasua belong to Lactobacillus, Staphylococcus and Escherichia groups (Fig. 4).

Relative abundance and microbial diversity.
The rank of bands and abundance curve showed the relationship between richness and evenness of OTU in each inasua community (Fig. 5). Shannon-Wiener diversity index (H') indicates that bacterial community diversity of inasua-S was 1.42 (medium), while inasua-NS was 0.90 (low). The evenness and dominance indices of both types of inasua were also different where evenness index of inasua-NS was 0.68 (high) and inasua-S was 0.52 (medium). The dominance index of inasua-NS was 0.52, whereas inasua-S 0.37 (Fig. 6).

DISCUSSION
The succession of Lactobacillus and Leuconostoc during fermentation was strongly influenced by the characteristics of inasua. The factor that greatly affected the diversity of lactic acid bacteria in inasua-NS was salt content. In contrast, in inasua-S,  Illustrations of DGGE banding patterns employing Phoretix 1D software (B) the limiting factors that affected the microbial diversity were acid and alcohol contents (13). LAB have varying tolerance to salinity, acid and alcohol. The amount of LAB in inasua-NS was lower than in inasua-S. Lactobacillus is one of the LAB that can grow in high salt, acid and alcohol as well as low oxygen conditions (14). Lactobacillus usually grows optimally at 5-6% salt content (15) and is tolerant to pH below 4.5 (16) and 4% alcohol content (17).
Lactobacillus plantarum is a dominant LAB in fish (18). L. plantarum has the ability to use several types of amino acids as substrates, making it capable of surviving in fish with relatively low carbohydrate content (19). Its optimum growth at 4-6% salt content and pH 4-9 conditions makes it easy to find it in various ecological niches (20). L. plantarum plays a role in various fermented fish products from Thailand, such as somfak and plasoom that have salt content of 3-5% (21). Other LAB found in fish is Lactobacillus rhamnosus (18). Some of the bacteri-  al strains have potential as probiotics (22). L. mesenteroides was found as dominant LAB at the end of inasua-NS fermentation. It is an obligate heterofermentative bacteria (23). Increasing pH at the end of inasua fermentation supported the growth of the LAB. L. mesenteroides and L. plantarum play roles in the production of shikae, a fermented fish product from Korea (24). After week 4 of fermentation, L. paracasei was found in inasua-S fermentation. Its presence was due to the decreasing alcohol content at end of fermentation (13). Lactobacillus paracasei has lower tolerance to alcohol than L. plantarum (23). Both play a role in the fermentation of coconut sap (25). At the end of inasua-S fermentation, L. plantarum was still the dominant LAB. In addition to being a dominant LAB in fish, L. plantarum is also dominant in fermented sap (26).
The results of DGGE showed that most of the bacteria found in inasua-NS was Staphylococcus. Addition of more than 9% salt in fish fermentation can suppress the growth of Lactobacillus that plays a role in fish fermentation and supports the growth of salt tolerant pathogenic bacteria (e.g. Staphylococcus) (27). High salt content can inhibit the growth of spoiling bacteria, but at the same time it slows down the rate of fermentation (28). Naturally, Staphylococcus epidermidis and S. pasteuri are absent in fish. Their presence in fermented fish products is due to human contact during the preparation as well as unhygienic processing (29). Low water activity and high salt content strongly support the growth of halophilic and halotolerant bacteria, including Staphylococcus in fish fermentation (30). S. epidermidis plays a role in various fermented fish products from Thailand, such as plara and nampla where salt content of which is 11-24% (21). L. curieae was found in both inasua-S and inasua-NS. The LAB lives in various environments. The presence of which in several fermented products with salt indicates that the LAB can adjust in high salt condition during the fermentation.
Numerous LAB were found dominating inasua-S fermentation. The LAB are from fish, sap and inasua processing. The diversity of LAB is influenced by the sources as well as salinity, acid and alcohol contents during fermentation. The salt content below 7% can increase the growth of LAB that plays an important role in fish fermentation (27). Low pH and high ethanol contents at the beginning of fermentation were the limiting factors for the growth of pathogenic and spoilage bacteria in inasua-S.
Lactobacillus apinorum, L. hilgardii, L. paracasei, L. nagelii and L. sucicola are LAB found in fermented sap. The dominance of the bacteria from coconut sap in inasua was due to the fact that coconut sap contains simple carbohydrates easily used by bacteria rather than complex carbohydrates in fish. Lactobacillus apinorum is commonly found in flowers and fruits with high sugar content. The bacteria, originally isolated from the honeybee stomach, is a fructophilic LAB that tends to use fructose rather than glucose as substrate for its growth (31). Coconut sap is a source of nutrients for microbial growth because it contains high fructose (32).
Lactobacillus hilgardii was also found in inasua-S fermentation. The LAB is often found in sap fermentation because it was resistant against high alcohol contents. L. hilgardii produces lactic acid and grows at an optimum pH below 4.5 (33). It is a heterofermentative LAB. The ability of L. hilgardii to produce alcohol and various organic compounds most likely adds inasua-S's sensory quality.
The other LAB found in inasua-S was Lactobacillus sucicola (sucus = sap). The LAB is capable of producing lactic acid through homofermentative pathways and is commonly found in fermented sap to produce traditional alcoholic beverages (34). Due to its tolerance to high alcohol and acid contents, L. nagelii grows in fermented sap (35). The last LAB found in inasua-S was L. paracasei that was also found in inasua-S fermentation with culture method since week 4 until the end of fermentation.
The uncontrolled processing of inasua allows the presence of microbial contaminants in fermentation. The presence of E. fergusonii in inasua-S was from non-aseptic sap tapping processor unhygienic inasua process. Most likely, it was from the equipment used in sap tapping (32). E. fergusonii can be found in low salt fish processing (36).
The OTU richness in inasua-NS (4 band) was lower than in inasua-S (8 band) due to high salt content in inasua-NS. The addition of coconut sap containing a number of microorganisms contributed to the high OTU richness in inasua-S. Dominance index of inasua-NS (0.52) was higher than inasua-S (0.37). Both types of inasua have one dominant OTU. L. apinorum and S. epidermidis have highest abundance of OTU in inasua-S and inasua-NS, respectively.

CONCLUSION
The succession of lactic acid bacteria during inasua fermentation is strongly influenced by physicochemical characteristics of inasua. Lactobacillus plantarum was found dominant during fermentation. At the end of inasua-S fermentation, L. plantarum was found dominant, while inasua-NS was dominated by Leuconostoc mesenteroides. Lactobacillus paracasei is a LAB found only in the inasua-S fermentation. The result of DGGE revealed that the dominant bacteria in inasua-NS was Staphylococcus, while in inasua-S was Lactobacillus. The bacterial diversity index in inasua-S was higher than in inasua-NS.